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A high-throughput imaging and nuclear segmentation analysis protocol for cleared 3D culture models
Imaging and subsequent segmentation analysis in three-dimensional (3D) culture models are complicated by the light scattering that occurs when collecting fluorescent signal through multiple cell and extracellular matrix layers. For 3D cell culture models to be usable for drug discovery, effective an...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6057966/ https://www.ncbi.nlm.nih.gov/pubmed/30042482 http://dx.doi.org/10.1038/s41598-018-29169-0 |
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author | Boutin, Molly E. Voss, Ty C. Titus, Steven A. Cruz-Gutierrez, Kennie Michael, Sam Ferrer, Marc |
author_facet | Boutin, Molly E. Voss, Ty C. Titus, Steven A. Cruz-Gutierrez, Kennie Michael, Sam Ferrer, Marc |
author_sort | Boutin, Molly E. |
collection | PubMed |
description | Imaging and subsequent segmentation analysis in three-dimensional (3D) culture models are complicated by the light scattering that occurs when collecting fluorescent signal through multiple cell and extracellular matrix layers. For 3D cell culture models to be usable for drug discovery, effective and efficient imaging and analysis protocols need to be developed that enable high-throughput data acquisition and quantitative analysis of fluorescent signal. Here we report the first high-throughput protocol for optical clearing of spheroids, fluorescent high-content confocal imaging, 3D nuclear segmentation, and post-segmentation analysis. We demonstrate nuclear segmentation in multiple cell types, with accurate identification of fluorescently-labeled subpopulations, and develop a metric to assess the ability of clearing to improve nuclear segmentation deep within the tissue. Ultimately this analysis pipeline allows for previously unattainable segmentation throughput of 3D culture models due to increased sample clarity and optimized batch-processing analysis. |
format | Online Article Text |
id | pubmed-6057966 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-60579662018-07-31 A high-throughput imaging and nuclear segmentation analysis protocol for cleared 3D culture models Boutin, Molly E. Voss, Ty C. Titus, Steven A. Cruz-Gutierrez, Kennie Michael, Sam Ferrer, Marc Sci Rep Article Imaging and subsequent segmentation analysis in three-dimensional (3D) culture models are complicated by the light scattering that occurs when collecting fluorescent signal through multiple cell and extracellular matrix layers. For 3D cell culture models to be usable for drug discovery, effective and efficient imaging and analysis protocols need to be developed that enable high-throughput data acquisition and quantitative analysis of fluorescent signal. Here we report the first high-throughput protocol for optical clearing of spheroids, fluorescent high-content confocal imaging, 3D nuclear segmentation, and post-segmentation analysis. We demonstrate nuclear segmentation in multiple cell types, with accurate identification of fluorescently-labeled subpopulations, and develop a metric to assess the ability of clearing to improve nuclear segmentation deep within the tissue. Ultimately this analysis pipeline allows for previously unattainable segmentation throughput of 3D culture models due to increased sample clarity and optimized batch-processing analysis. Nature Publishing Group UK 2018-07-24 /pmc/articles/PMC6057966/ /pubmed/30042482 http://dx.doi.org/10.1038/s41598-018-29169-0 Text en © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article Boutin, Molly E. Voss, Ty C. Titus, Steven A. Cruz-Gutierrez, Kennie Michael, Sam Ferrer, Marc A high-throughput imaging and nuclear segmentation analysis protocol for cleared 3D culture models |
title | A high-throughput imaging and nuclear segmentation analysis protocol for cleared 3D culture models |
title_full | A high-throughput imaging and nuclear segmentation analysis protocol for cleared 3D culture models |
title_fullStr | A high-throughput imaging and nuclear segmentation analysis protocol for cleared 3D culture models |
title_full_unstemmed | A high-throughput imaging and nuclear segmentation analysis protocol for cleared 3D culture models |
title_short | A high-throughput imaging and nuclear segmentation analysis protocol for cleared 3D culture models |
title_sort | high-throughput imaging and nuclear segmentation analysis protocol for cleared 3d culture models |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6057966/ https://www.ncbi.nlm.nih.gov/pubmed/30042482 http://dx.doi.org/10.1038/s41598-018-29169-0 |
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